The Promokine Bioluminescent Cell Viability Kit I employs bioluminescence to measure the ATP level as a marker for cell viability in mammalian cells. It utilizes the enzyme luciferase to catalyze the ATP-dependent, light-producing oxidation of luciferin. This light formation can be measured using a luminometer or beta counter.
The assay can be performed directly in culture plates requiring no harvest, washing, or sample preparation and can be fully automated for high throughput (10 seconds/sample). It is highly sensitive, detecting 10-100 mammalian cells/well, and it also offers new applications besides cell viability testing, e.g., detecting low level bacterial contamination in blood, milk, urine, soil, and sludge.

At PromoCell we guarantee for our primary human cells ≥ 500,000 viable cells after thawing. For this, we dispense > 500,000 cells per cryovial before cryopreservation as there will always be a certain percentage of dead cells after freeze/thaw.
In order to know the number of cells that survived the procedure, we defrost a representative number of vials per lot during QC, determine the cell viability using an electronic counting device and then calculate the number of viable cells that can be recovered after thawing. Both numbers – the calculated number of viable cells and the viability – can be found on the lot-specific Certificate of Analysis (CoA) that can be downloaded from our website.Example: When the CoA indicates 600,000 viable cells and a viability of 80%, this means that the vial actually contains 750,000 cells (viable + dead), 80% thereof (600,000) were viable after thawing in our QC. We do not indicate the total number of cells per vial but just the number of expected viable cells which can be recovered when the recommended thawing protocol is used. You don’t have to calculate any viabilities by yourself.When the recommended plating density for your cell type is 5,000 – 10,000 cells/cm², then the 600,000 viable cells can be plated e.g. in a T75 (corresponding to 8,000 cells/cm²) or in a T75 + a T25 (corresponding to 6,000 cells/cm²).

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We have tailored and optimized a protocol for suspension cells that is detailed in the Instruction Manual. Briefly, during the incubation step when MATra reagent/nucleic acid complexes or MA Lipofection Enhancer/nucleic acid/transfection reagent (or virus) complexes will form, dilute the cells to be transfected to 5×105 – 1×106 per ml in medium (with or without serum or supplement) and perform one of the following options to sediment the cells at the bottom of the culture dish thus making them susceptible to MATra:

Option 1: Mix cell suspension with 30 µl of MATra-S Immobilizer per 1 ml of cell suspension.a. Incubate for 10-15 minutes.b. Distribute cells to the culture dish placed upon the magnet plate (volume of culture medium containing cells depends on the culture dish size; see transfection volume as suggested in the protocol).c. Incubate for 15 minutes.d. Add the mixture containing the nucleic acid/MATra reagent complexes to the cells while keeping the culture plate on the magnet plate, continue to incubate for 15 minutes and then proceed as explained in the Instruction Manual.

Option 2: Seed the cells on polylysine-coated plates and continue with the protocol for adherent cells (see Instruction Manual).

Option 3: Briefly centrifuge the cells (2 minutes) in order to pellet them and continue with the protocol for adherent cells (see Instruction Manual).

Option 4: Use antibody against a cell surface antigen immobilized on the microplate and continue with the protocol for adherent cells (see Instruction Manual).

Annexin V-Cy3 Detection Kit and Annexin V-PE Detection Kit include the conjugated Annexin V and 1x Binding Buffer. They can be used to quantify the ratio of apoptotic (red/orange fluorescence) versus non-apoptotic (non-fluorescent) cells.
The Annexin V-Cy3/ -PE Detection KitsPlus additionally include SYTOX green dye, which is impermeable to live and apoptotic cells, but stains necrotic cells by binding to nucleic acids. After staining a cell population with these "Plus" Kits, apoptotic cells show red fluorescence, dead cells show green fluorescence and live cells show little or no fluorescence. The populations can easily be distinguished by fluorescence microscopy using FITC and rhodamine filters or by flow cytometry using the FL1 channel (Ex. 488 nm/Em. 530 nm) for SYTOX Green dye and FL2 channel for Annexin V-Cy3 or -PE (Ex. 543 nm/Em. 570 nm).

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For adherent cells, at least 1,000 cells are necessary per well (100 µl medium) when using a standard 96-well plate. For leukocytes and other suspension cells, at least 2,500 cells/well (100 µl medium) are necessary because of lower sensitivity. The recommended maximum number of cells per well for the 96-well plate is 25,000. For 384 well plates, just divide these cell numbers by 4 (250 cells per well for adherent, 625 cells per well for suspension cells). If 24-well or 6-well plates are used for the assay, please calculate the number of cells per well accordingly, and adjust the volume of the WST-8 solution in each well to 10% of the total volume. For more exotic culture systems, like 3-D models or sphereoids, you should start with 10% WST-8 reagent per vol. of media and culture until you get a good reading. Some optimization of the protocol may then be necessary because the cells‘ metabolisms behave differently in 3-D.

It is recommended to measure the background wavelength; however, the background should be very low compared to the signal. Measuring the background confirms that there is no unexpected signal (for example, from the plate itself) that might interfere with the reading.

Our Live/Dead Cell Staining Kit is for use in unfixed living cells, so it cannot be used in paraffin sections or cryosections of tissue.
There are some references reporting the use of calcein-AM and vital dyes in dissected tissues or organ cultures in vitro. The main concern would be whether the dyes can penetrate the areas of tissue that you want to examine. Also, calcein is not fixable, so you would need to observe the fluorescence in the tissue without additional processing. A longer incubation time might be required in tissue specimens compared to cells to allow the dyes to penetrate and for conversion calcein-AM into calcein. Also, because ethidium homodimer III stains cell with compromised membrane integrity, it will likely stain cells along cut edges or on the surface of a tissue slice.
A fixable alternative to calcein-AM is CFSE, which is non-fluorescent until it is hydrolyzed by esterases in the cytoplasm of living cells to generate fluorescein-SE, which covalently attaches to cellular proteins. We offer CFSE (Cell Proliferation Kit I, PK-CA707-30050), which you could pair with a vital dye like propidium iodide or ethdium homodimer III to stain dead cells. For the vital dyes, it is important to wash away the free dye thoroughly before fixing to avoid background staining.

Our Annexin V Detection Kits are all based on the fact that apoptotic cells translocate the membrane phosphatidyl-serine (PS) from the inner surface of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by means of Annexin V, a protein that has a very strong affinity for PS. To visualize the PS/Annexin V binding, Annexin V is coupled to a fluorescent dye. PromoKine supplies a variety of different fluorescent dyes (with different Excitation/Emission characteristics) to allow the end user to choose the kit that is best suited for his/her research, e.g. depending on the filters your fluorescent microscope is equipped with.
Cy3 and Cy5 are two fluorescent dyes that are commonly used for cell analyses. The characteristics of these dyes are as follows:Cy3: Ex = 543 nm; Em = 570 nm; apoptotic cells that have bound Annexin V-Cy3 will show red staining of the plasma membrane. Cy5: Ex = 649 nm; Em = 670 nm; apoptotic cells that have bound Annexin V-Cy5 will show bright red-blue staining of the plasma membrane.
In other words, the fluorescent dye (e.g. Cy3, Cy5) determines the color of plasma membrane staining. The kit should however work with mononuclear cells irrespective of the conjugated dye.

The PromoCell specialized media are optimized for in vitro-culture of human cells. In most cases, they can also be used for cells from several other species (eg. rat, murine, bovine, porcine cells) and etablished cell lines.
Please see the attached document for references that have used our media for other cell types and species.

You may wish to process floating and detached cells separately – collect the washes and spin them down and then stain those cells using the protocol for suspension cells.
Alternatively you may collect floating cells, detach the adherent cells with trypsin (without EDTA), pool the cells together, and use the suspension cell protocol for staining.

PromoCell does not recommend a split ratio for Normal Human Cells because the cell yield varies from lab to lab. We recommend to count the cells after trypsinization and to replate them at 5,000-10,000 cells/cm². The precise recommended plating density for each cell type can be found in the Instruction Manual.

“Normal” means that the cells in question were freshly isolated from non-tumour tissue. They have a finite life span in vitro. They have neither been genetically modified nor treated and/or selected to produce a continous cell line. “Normal” cells are primary cells that have been subcultured at least once.

There is no general recommendation regarding the passage number at which to perform your experiments. This depends on the cell type as well as on the type of experiment. Most scientists don’t exceed passage 4 to 6 when using normal cells for their experiments as the cells gradually change their phenotype in vitro. If a certain phenotype is essential for your experiments, you should run expression controls at regular intervals to ensure that the markers are still sufficiently expressed in your cells.

The PromoCell media are optimized for in vitro-culture of human cells. In most cases, they can also be used for cells from several other species (eg. rat, mouse, bovine, porcine cells). To view a list of references where our media were used with other species/cell types please click here.

All cells are shipped from our premises in Germany. The cryopreserved vials are shipped with plenty of dry ice. For some countries, when delivery cannot be guaranteed within 3-4 days, we use vapor shippers. Upon receipt of the cells, store them in liquid nitrogen until they are thawed and seeded. We have shipped many cryopreserved cells to countries all over the world. When they are thawed according to the PromoCell protocol, viabilities are achieved as specified in the CoA (≥70%).

Unless otherwise stated, PromoCell Human Cells are derived from single donors. The donor’s age, sex, and race are specified in the lot-specific Certificate of Analysis which can be downloaded here. For a few selected cell types, we also supply cells from pooled donors. These include HUVEC pooled (# C-12203/C-12253; # C-12208/C-12209) and NHEK pooled (# C-12005/C-12007; C-12006/C-12008).

Proliferating cells are shipped in T25 or T75 flasks completely filled with Growth Medium together with warm packs (37°C) that keeps the cells warm for several hours during the transport. Usually, the flasks arrive at room temperature and should be placed in the incubator for 3 hrs before changing the media or subculturing the cells.
Please note: Proliferating cultures are only shipped to those countries where a 24 h delivery can be guaranteed.

PromoCell Normal Human Cells should be cultured at 37°C and 5% CO2 in a humidified atmosphere.
Please note: If using cell culture flasks w/o filter cap, unscrew the cap by half a turn to allow for sufficient ventilation.

For optimal cell growth, we recommend to refrain from using antibiotics. Most primary or normal human cells show reduced growth rates in the presence of antibiotics. In some cases, when a sterile environment cannot be 100% ensured, it may be advisable to add antibiotics to the media to protect the culture from potential microbial infections.

Antibiotics are routinely used in cell cultures to prevent bacterial infections. But there are side effects: Studies show that they impair cell growth and differentiation. With good laboratory practice, the use of antibiotics is unnecessary.
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